Project Summary The broad objectives are 1) to determine the mechanisms by which protein stability is achieved, and in particular, how stabilities of distant segments of proteins influence each other, giving rise to cooperativity, using a combination of natural and designed repeat proteins, and 2) to understand and leverage phylogenic-based consensus approaches to design proteins for high stability and high levels of activity. These objectives will be achieved through three specific aims. 1) Apply our nearest-neighbor 1D Ising formalism that we developed for natural repeat proteins to quantify local folding and nearest neighbor coupling energies to a series of de novo designed helical repeat proteins from the Baker lab. In parallel, we will measure folding kinetics, using the energy landscape framework that results from the Ising analysis as a framework for interpretation. Comparison to natural repeat proteins will reveal differences in folding between designed and natural proteins. 2) Apply consensus design methods that we have used to stabilize linear repeat proteins to globular proteins of different folds, sizes, and functions, and 3) determine the extent to which biological activity is maintained. In Aim 2, we have identified sixteen targets, and have strong preliminary results for six. We will determine structures by NMR and x-ray crystallography, and stabilities using solution thermodynamics and kinetics. We will dissect the basis of increased stability using sequence and structure metrics, and compare with the ancestral reconstruction approach. In Aim 3, we will measure binding affinities, specificities, and enzyme activities, and will focus on whether high stabilities decrease activity, and whether dynamics changes is a general correlate. All three of these aims will use large numbers of comparisons among different proteins to build a statistically rigorous and general picture of design and consensus features, allowing us to generalize, determining what works, what does not, and why. This is a significant improvement over the one-off anecdotal studies that have been described to date. Achieving these objectives will advance our understanding of the constraints on naturally occurring protein sequences and evolution, and will address several paradigms including the principle of minimal frustration and the stability-activity tradeoff, and will identify key differences between de novo-designed and natural protein sequences. These studies will provide a deeper and more complete understanding of protein folding, and will also improve our ability to design proteins for biotechnology and medicine.